The purpose of this project is to develop techniques for the isolation of native Factor V in sufficient quantity and purity to allow detailed studies on the subunit structure and to facilitate studies on mechanism of action of Factor V. Following isolation and characterization of the subunit structure of Factor V, chemical characterization of the thrombin catalyzed activation will be pursued. Activation peptides, if any, will be isolated and their possible biological activities studied. Once Factor V is purified, (1) the role of the Ca ions in maintaining biological activity will be investigated in both native and activated Factor V, (2) the binding of Factor V and Factor V subunits to phospholipid in the presence and absence of Ca ions will be studied and (3) the phospholipid binding fragment (Pro Fragment 1) from prothrombin will be tested to determine if it can displace Factor V from the phospholipid in a manner analogous to its displacement of prothrombin. The mechanistic details of Factor V catalysis of prothrombin activation will then be investigated kinetically by employing purified Factor Xa, prothrombin and prothrombin activation fragments and intermediates. Kinetic evidence will be sought to prove or disprove the hypothesis that Factor V must be activated by thrombin before it can function in prothrombin activation.